Visualization of Nuclease- and Serum-Mediated Chromatin Degradation with DNA-Histone Mesostructures.

This study analyzed the nuclease- and serum-driven degradation of millimeter-scale, circular DNA-histone mesostructures (DHMs). DHMs are bioengineered chromatin meshes of defined DNA and histone compositions designed as minimal mimetics of physiological extracellular chromatin structures, such as neutrophil extracellular traps (NETs). Taking advantage of the defined circular shape of the DHMs, an automated time-lapse imaging and image analysis method was developed and used to track DHM degradation and shape changes over time. DHMs were degraded well by 10 U/mL concentrations of deoxyribonuclease I (DNase I) but not by the same level of micrococcal nuclease (MNase), whereas NETs were degraded well by both nucleases. These comparative observations suggest that DHMs have a less accessible chromatin structure compared to NETs. DHMs were degraded by normal human serum, although at a slower rate than NETs. Interestingly, time-lapse images of DHMs revealed qualitative differences in the serum-mediated degradation process compared to that mediated by DNase I. Importantly, despite their reduced susceptibility to degradation and compositional simplicity, the DHMs mimicked NETs in being degraded to a greater extent by normal donor serum compared to serum from a lupus patient with high disease activity. These methods and insights are envisioned to guide the future development and expanded use of DHMs, beyond the previously reported antibacterial and immunostimulatory analyses, to extracellular chromatin-related pathophysiological and diagnostic studies.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

Extracellular Trap-Mimicking DNA-Histone Mesostructures Synergistically Activate Dendritic Cells.

Extracellular traps (ETs), such as neutrophil extracellular traps, are a physical mesh deployed by immune cells to entrap and constrain pathogens. ETs are immunogenic structures composed of DNA, histones, and an array of variable protein and peptide components. While much attention has been paid to the multifaceted function of these structures, mechanistic studies of ETs remain challenging due to their heterogeneity and complexity. Here, a novel DNA-histone mesostructure (DHM) formed by complexation of DNA and histones into a fibrous mesh is reported. DHMs mirror the DNA-histone structural frame of ETs and offer a facile platform for cell culture studies. It is shown that DHMs are potent activators of dendritic cells and identify both the methylation state of DHMs and physical interaction between dendritic cells and DHMs as key tuning switches for immune stimulation. Overall, the DHM platform provides a new opportunity to study the role of ETs in immune activation and pathophysiology.

Antimicrobial Microwebs of DNA-Histone Inspired from Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are decondensed chromatin networks released by neutrophils that can trap and kill pathogens but can also paradoxically promote biofilms. The mechanism of NET functions remains ambiguous, at least in part, due to their complex and variable compositions. To unravel the antimicrobial performance of NETs, a minimalistic NET-like synthetic structure, termed "microwebs," is produced by the sonochemical complexation of DNA and histone. The prepared microwebs have structural similarity to NETs at the nanometer to micrometer dimensions but with well-defined molecular compositions. Microwebs prepared with different DNA to histone ratios show that microwebs trap pathogenic Escherichia coli in a manner similar to NETs when the zeta potential of the microwebs is positive. The DNA nanofiber networks and the bactericidal histone constituting the microwebs inhibit the growth of E. coli. Moreover, microwebs work synergistically with colistin sulfate, a common and a last-resort antibiotic, by targeting the cell envelope of pathogenic bacteria. The synthesis of microwebs enables mechanistic studies not possible with NETs, and it opens new possibilities for constructing biomimetic bacterial microenvironments to better understand and predict physiological pathogen responses.

Nanoparticle Assemblies into Luminescent Dendrites in Shrinking Microdroplets.

The self-assembly of nanoparticles (NPs) is essential for emerging dispersion-based energy-conscious technologies. Of particular interest are micro- and macro-scale self-organizing superstructures that can bridge 2D/3D processing scales. Here we report the spontaneous assembly of CdTe NPs within an aqueous microdroplet suspended in soybean oil. The gradual diffusion of the water into the surrounding medium results in shrinking of the microdroplet, and a concomitant formation of branched assemblies from CdTe NPs that evolve in size from ∼50 µm to ∼1000 µm. The fractal dimension of NP assemblies increases from ∼1.7 to ∼1.9 during the assembly process. We found that constituents of the soybean oil enter the aqueous solution across the microdroplet interface and affect NP assembly. The obtained NP dendrites can be further altered morphologically by illumination with light that results in the disassembly of the NP dendrites. The use of this microheterogeneous dispersion platform with partially soluble hydrophilic and hydrophobic solvents highlights the sensitivity of the NP assembly process to environment and presents an opportunity to explore droplet-confined NP assembly.

Fracture fabrication of a multi-scale channel device that efficiently captures and linearizes DNA from dilute solutions.

This paper describes a simple technique for patterning channels on elastomeric substrates, at two distinct scales of depth, through the use of controlled fracture. Control of channel depth is achieved by the careful use of different layers of PDMS, where the thickness and material properties of each layer, as well as the position of the layers relative to one another, dictate the depth of the channels formed. The system created in this work consists of a single 'deep' channel, whose width can be adjusted between the micron- and the nano-scale by the controlled application or removal of a uniaxial strain, and an array of 'shallow' nano-scale channels oriented perpendicular to the 'deep' channel. The utility of this system is demonstrated through the successful capture and linearization of DNA from a dilute solution by executing a two-step 'concentrate-then-linearize' procedure. When the 'deep' channel is in its open state and a voltage is applied across the channel network, an overlapping electric double layer forms within the 'shallow' channel array. This overlapping electric double layer was used to prevent passage of DNA into the 'shallow' channels when the DNA molecules migrate into the junctional region by electrophoresis. Release of the applied strain then allows the 'deep' channel to return to its closed state, reducing the cross-sectional area of this channel from the micro- to the nano-scale. The resulting hydrodynamic flow and nano-confinement effects then combine to efficiently uncoil and trap the DNA in its linearized form. By adopting this strategy, we were able to overcome the entropic barriers associated with capturing and linearizing DNA derived from a dilute solution.